ORIGINAL RESEARCH ARTICLE published: 29 May 2013 doi: 10.3389/fmicb.2013.00084 Metagenome sequence analysis of filamentous microbial communities obtained from geochemically distinct geothermal channels reveals specialization of three Aquificales lineages CristinaTakacs-Vesbach1*,William P.Inskeep 2*, Zackary J. Jay 2, Markus J. Herrgard 3, Douglas B. Rusch4, Susannah G.Tringe 5, Mark A. Kozubal 2, Natsuko Hamamura6, Richard E. Macur 2, Bruce W. Fouke 7, Anna-Louise Reysenbach8,Timothy R. McDermott 2, Ryan deM. Jennings 2, Nicolas W. Hengartner 9 and Gary Xie 10 1 Department of Biology, University of New Mexico, Albuquerque, NM, USA 2 Department of Land Resources and Environmental Sciences, Thermal Biology Institute, Montana State University, Bozeman, MT, USA 3 Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Hørsholm, Denmark 4 Center for Genomics and Bioinformatics, Indiana University, Bloomington, IN, USA 5 Department of Energy-Joint Genome Institute, Walnut Creek, CA, USA 6 Center for Marine Environmental Studies, Ehime University, Matsuyama, Ehime, Japan 7 Roy J. Carver Biotechnology Center, University of Illinois, Urbana, IL, USA 8 Department of Biology, Portland State University, Portland, OR, USA 9 Computer, Computational, and Statistical Sciences Division, Los Alamos National Laboratory, Los Alamos, NM, USA 10 Bioscience Division, Los Alamos National Laboratory, Los Alamos, NM, USA Edited by: The Aquificales are thermophilic microorganisms that inhabit hydrothermal systems world- Martin G. Klotz, University of North wide and are considered one of the earliest lineages of the domain Bacteria. We analyzed Carolina at Charlotte, USA metagenome sequence obtained from six thermal “filamentous streamer” communities Reviewed by: Olivia Mason, Florida State University, (s40 Mbp per site), which targeted three different groups of Aquificales found in Yellow- USA stone National Park (YNP). Unassembled metagenome sequence and PCR-amplified 16S Martin Keller, Oak Ridge National rRNA gene libraries revealed that acidic, sulfidic sites were dominated by Hydrogenobac- Laboratory, USA ulum (Aquificaceae) populations, whereas the circum-neutral pH (6.5–7.8)sites containing *Correspondence: dissolved sulfide were dominated by Sulfurihydrogenibium spp. (Hydrogenothermaceae). Cristina Takacs-Vesbach, Department of Biology, University of New Mexico, Thermocrinis (Aquificaceae) populations were found primarily in the circum-neutral sites MSC03 2020, Albuquerque, NM with undetectable sulfide, and to a lesser extent in one sulfidic system at pH 8. Phyloge- 87131, USA. netic analysis of assembled sequence containing 16S rRNA genes as well as conserved e-mail: [email protected]; protein-encoding genes revealed that the composition and function of these communi- William P.Inskeep, Department of Land Resources and Environmental ties varied across geochemical conditions. Each Aquificales lineage contained genes for Sciences, Thermal Biology Institute, CO2 fixation by the reverse-TCA cycle, but only the Sulfurihydrogenibium populations per- Montana State University, Bozeman, form citrate cleavage using ATP citrate lyase (Acl). The Aquificaceae populations use an MT 59717, USA. alternative pathway catalyzed by two separate enzymes, citryl-CoA synthetase (Ccs), and e-mail: [email protected] citryl-CoA lyase (Ccl). All three Aquificales lineages contained evidence of aerobic respira- tion, albeit due to completely different types of heme Cu oxidases (subunit I) involved in oxygen reduction. The distribution of Aquificales populations and differences among func- tional genes involved in energy generation and electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, H2,O2) have resulted in niche specialization among members of the Aquificales. Keywords: thermophiles, functional genomics, phylogeny, autotrophic processes, sulfide oxidation INTRODUCTION Boussau et al., 2008; Zhaxybayeva et al., 2009). The Aquificales The order Aquificales represents a group of thermophilic microor- include two predominant families, the Hydrogenothermaceae and ganisms that inhabit marine and terrestrial hydrothermal sys- Aquificaceae, and both are well-represented in different geother- tems worldwide (Ferrera et al., 2007). This lineage is of sig- mal features of Yellowstone National Park (YNP) (Reysenbach nificant interest because its members are believed to comprise et al., 2005). Members of the Hydrogenothermaceae include the the deepest lineage of the domain Bacteria (Coenye and Van- genus Sulfurihydrogenibium, which inhabit circum-neutral sul- damme, 2004; Barion et al., 2007), although alternative evolu- fidic springs in YNP (Hugenholtz et al., 1998; Reysenbach et al., tionary histories have been suggested (Griffiths and Gupta, 2004; 2000b, 2005). The Aquificaceae comprise two divergent groups: www.frontiersin.org May 2013 | Volume 4 | Article 84 | 1 Takacs-Vesbach et al. Metagenome analysis of Aquificales microbial communities Hydrogenobaculum spp. are found predominantly in low-pH sys- 2008). Additionally, the abundance of molecular hydrogen in tems (pH < 4) (Jackson et al., 2001; Langner et al., 2001; Macur hydrothermal systems was taken as evidence for the predomi- et al., 2004; D’Imperio et al., 2007, 2008; Hamamura et al., 2009), nance of this metabolism in thermal features from YNP (Spear while Thermocrinis-like organisms generally exhibit higher-pH et al., 2005). However, the recent isolation of several new terres- ranges (pH 6–9), overlapping with the near circum-neutral opti- trial species indicates some organisms within this group are unable mum for Sulfurihydrogenibium (Reysenbach et al., 1994; Blank to grow on hydrogen and do not contain Group I Ni-Fe hydro- et al., 2002). genases (Reysenbach et al., 2009). Diverse metabolisms have been Given our limited understanding of their phylogenetic history detected among cultured members of the Aquificales including the and metabolic potential, numerous questions remain regarding use of H2, elemental sulfur, and thiosulfate as energy sources, and the ecology and evolution of the Aquificales. For example, the although generally aerobic, some members are microaerophilic Aquificales are important primary producers in hydrothermal sys- and/or utilize nitrate as an electron acceptor (D’Imperio et al., tems (Harmsen et al., 1997; Yamamoto et al., 1998; Reysenbach 2008; Reysenbach et al., 2009). Thus, while the metabolic poten- et al., 1999, 2000a; Blank et al., 2002; Eder and Huber, 2002; Ina- tial within the Aquificales is largely known from a few well-studied gaki et al., 2003; Spear et al., 2005) and several members of this isolates, there is not an equal distribution of representatives from lineage have been shown to use the reductive tricarboxylic acid (r- each of the families in culture, and their physiological capabilities TCA) cycle for the fixation of carbon dioxide (Shiba et al., 1985; remain unknown, especially under the environmental conditions Beh et al., 1993; Ferrera et al., 2007). However, several members that are characteristic of their natural habitats. of the order are also capable of heterotrophy (Huber et al., 1998; Much of what is known about the distribution of the Aquifi- Nakagawa et al., 2005; Caldwell et al., 2010), which makes their cales is based on molecular diversity studies across different habitat ecological role as possible primary producers unclear. Moreover, types (Reysenbach et al., 1994, 2000b; Hugenholtz et al., 1998; the diversity and variation of r-TCA-specific enzymes and sub- Stohr et al., 2001; Van Dover et al., 2001; Inagaki et al., 2003), strates used by cultured members has led to speculation regarding although their ecology has been inferred largely from the geo- the origin and the distribution of this pathway within the phylum chemical conditions they inhabit (Fouke et al., 2000; Spear et al., (Hugler et al., 2007). 2005; Hall et al., 2008; Hamamura et al., 2009) and the physi- The Aquificales were originally named for the ability of the ology of the few cultivated members (Jahnke et al., 2001; Takai type strain, Aquifex pyrophilus, to oxidize molecular hydrogen et al., 2002; Reysenbach et al., 2009). Based on molecular diversity to water (Huber et al., 1992). Consequently, hydrogen oxidation surveys of 16S rRNA and metabolic genes, different Aquificales was generalized to the entire order based on the phenotype of a lineages do not generally share the same habitats in YNP (Rey- few cultivated members (Reysenbach and Cady, 2001; Donahoe- senbach et al., 2005), although exceptions have been noted where Christiansen et al., 2004; Huber and Eder, 2006; D’Imperio et al., Thermocrinis- and Sulfurihydrogenibium-like organisms have been FIGURE 1 | Site photographs of Aquificales “streamer” communities visually [DS_9 (elemental sulfur); OSP_14 (Fe-oxides); MHS_10 (calcium sampled fromYellowstone National Park. The sites represent diverse carbonate); CS_12 (pyrite, amorphous Fe-sulfides); OS_11 (none); BCH_13 geochemical environments as noted from the different mineralogy apparent (none, Fe(III)-staining on silica)]. Frontiers in Microbiology | Microbial Physiology and Metabolism May 2013 | Volume 4 | Article 84| 2 Takacs-Vesbach et al. Metagenome analysis of Aquificales microbial communities found together in sulfidic circum-neutral pH channels (Hall et al., sulfur due to both biotic and abiotic oxidation (D’Imperio et al., 2008; Hamamura et al., 2009). Therefore, the distinct separa- 2008), whereas lower concentrations of H2S(aq)